COPI domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP
COPI domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP
复制标题
DOI：
10.1093/emboj/19.15.3905
复制
发表时间：
2000-08-01
期刊：
影响因子：
11.4
通讯作者：
Duden, R
中科院分区：
文献类型：
--
作者：
Eugster, A;Frigerio, G;Duden, R
我们对CoP I亚基上的相互作用结构域进行了系统的映射，以获得对辅助剂结构的新见解，使用双杂交系统，我们表征了α、β‘、epsilon-COP和β-、伽马、三角洲、Zeta-COP辅助剂亚复合体的结构域结构，并确定了它们之间的联系，有助于辅助剂的完整性。我们的结果表明，β-、伽马-、β-、Zeta-COP亚复合体和AP适配器复合体的结构域组织是相关的。通过对α-COP截断突变体的体内分析，我们确定了α-COP上不同的功能结构域，其N-末端WD40结构域对于酵母细胞的活性和整体辅酶活性是必不可少的，但对于KKXX依赖的运输是必需的。最后类似于170个氨基酸的α-COP对细胞活力也不是必需的，但对于epsilon-COP掺入辅助体和维持正常的epsilon-COP水平是必需的。此外，我们还证明了辅酶亚基与调控蛋白的直接相互作用：β‘-和γ-COP与ARF-GTP激活蛋白(GAP)Glo3p相互作用，而不是Gcs1p，β-和epsilon-COP与ARF-GTP相互作用。Glo3p在体外也能与完整的辅酶原相互作用。
We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer, Using the two-hybrid system, we characterize the domain structure of the alpha-, beta'-, epsilon-COP and beta-, gamma-, delta-, zeta-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the beta-, gamma-, delta-, zeta-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of alpha-COP truncation mutants, we characterize distinct functional domains on alpha-COP, Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent trafficking. The last similar to 170 amino acids of alpha-COP are also nonessential for cell viability, but required for epsilon-COP incorporation into coatomer and maintainance of normal epsilon-COP levels. Further, we demonstrate navel direct interactions of coatomer subunits with regulatory proteins: beta'- and gamma-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and beta- and epsilon-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.